THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Cardiac ischemia is the leading cause of death in the developed nations. It has recently been established that an increase in the level of the hsp70 heat shock proteins in heart cells, such as myocytes, leads to a protective effect toward ischemia and other cellular stress. Clinical studies indicate that the presence of elevated levels of hsp70 can lead to a significant decrease in mortality after cardiac ischemia. The mechanism by which hsp70 exerts its cytoprotective function is not well understood: the proposed experiments will increase our understanding of the molecular basis-for the heat shock protein protection, and should lay the groundwork for subsequent therapeutic regimes. The current thinking is that ischemia or heat shock leads to denatured proteins which are then bound by hsp70, and that this results in cytoprotection, either by decreasing the amount of aggregated protein or by facilitating the subsequent recovery, or perhaps both. However, there are a number of observations which are not readily accounted for by this model, but which would be explained if hsp70 itself aggregated under these conditions. Our experiments are aimed at testing these hypotheses. Using a myocyte cell line, we will characterize the proteins aggregated during simulated ischemia and heat shock and determine the stoichiometry between hsp70 and other aggregated proteins. We will determine if the aggregated proteins are native or denatured, and whether they are complexed with hsp70 or physically entrapped. We will investigate whether changes in pH, and nucleotide and cation levels, may be responsible for the observed stress- induced aggregation of hsp70. We will also investigate the putative role of heat shock proteins and ATP in the resolubilization of stress-induced aggregates. We expect to determine the molecular basis for the aggregation of proteins in ischemic (ATP-depleted) cells, and to ascertain how similar it is to that for thermally-induced aggregation. Our proposed investigation should also provide more knowledge about the involvement and molecular role(s) of hsp40 and hsp90 in the stress response. Among the novel approaches we will use are computerized image analysis of 2D gels, and ATR FTIR spectroscopy to evaluate protein conformation in insoluble aggregates.